In recent years, abnormal methylation of DNA has been found to be deeply involved in malignant transformation and has received attention. Abnormal DNA methylation of CpG islands in some gene promoter regions is known as a characteristic epigenetic abnormality in cancer cells. The CpG island is a region in which a two-nucleotide sequence of cytosine (C)-guanine (G) via a phosphodiester bond (p) appears with high frequency. This region often resides in a promoter region upstream of a gene. The abnormal DNA methylation of the CpG island is involved in carcinogenesis through the inactivation of tumor suppressor genes, etc. DNA hypermethylation of the CpG island correlating with clinicopathological factors has been reported in colorectal cancer, stomach cancer, etc. (Non Patent Literatures 1 to 4). Such a type of cancer is called CpG island methylation phenotype (CIMP)-positive cancer.
Already established methods for analyzing methylated DNA include a method based on bisulfite reaction. This method is a method most generally used in the analysis of methylated DNA. The treatment of single-stranded DNA with bisulfite converts cytosine to uracil through sulfonation, hydrolic deamination, and desulfonation. On the other hand, methylated cytosine is left unaltered throughout the reaction time of actually performed bisulfite treatment because the reaction rate of sulfonation as the first step is very slow. Thus, PCR (polymerase chain reaction) using the bisulfite-treated DNA amplifies unmethylated cytosine with the uracil replaced with thymine, while leaving the methylated cytosine unaltered. The methylation status is analyzed through the use of the difference between the bases cytosine and thymine appearing in the sequence of this PCR amplification product. Methods generally used according to this basic principle are methylation-specific PCR (MSP) described in Patent Literature 1 and Non Patent Literature 5, and combined bisulfite restriction analysis (COBRA) described in Non Patent Literatures 6 and 7.
The MSP method is a method which involves sequentially performing PCR amplification using a methylated sequence-specific primer and an unmethylated sequence-specific primer and agarose gel electrophoresis after bisulfite treatment of DNA, and determining the DNA methylation status of the target region from the presence or absence of an amplification product derived from both of the primers. The COBRA method is a method which involves sequentially performing PCR amplification using common primers for methylated DNA and unmethylated DNA, treatment using a restriction enzyme recognizing a site differing in sequence between methylated DNA and unmethylated DNA, and agarose gel electrophoresis after bisulfite treatment of DNA, and determining the DNA methylation status of the target region from the presence or absence of the restriction enzyme-treated fragment. Both of these methods are methylated DNA analysis methods currently used widely because these methods are capable of quantitatively analyzing methylated DNA without special equipment. A problem of the methods, however, is time and labor required for electrophoresis used in the analysis.
Meanwhile, ion exchange chromatography is generally used as a method capable of convenient and accurate detection in a short time in the separation and analysis of biomacromolecules such as nucleic acids, proteins, and polysaccharides, for example, in biochemical and medical fields. In the case of separating a PCR amplification product of a nucleic acid by use of ion exchange chromatography, anion exchange chromatography, which separates the PCR amplification product through the use of the negative charge of phosphate contained in the nucleic acid molecule, is generally used. Columns for anion exchange chromatography packed with column packing materials having cationic functional groups as ion exchange groups have already been commercially available and used in various research fields.
It has been further reported that the single-nucleotide difference between 20-mer unmethylated synthetic oligonucleotides can be separated and analyzed by ion exchange chromatography using a column packed with a column packing material having both a strong cationic group and a weak cationic group as cationic functional groups (Patent Literature 2).
Renal cell carcinoma (RCC) often develops even in middle-aged people belonging to the working population. A great majority of RCC case groups are completely cured by nephrectomy, whereas case groups which progress rapidly into distal metastasis are obviously present. These curable and metastatic RCC case groups largely differ in their clinical courses. Furthermore, some of cases with metastasis are known to respond to immunotherapy, molecular targeting therapeutic drugs, or the like. There is the possibility that the prognosis of cases likely to have recurrence can be improved by close follow-up, early diagnosis of recurrence, and additional aftercare. However, some cases experience rapid distal metastasis of clear cell RCC even having a low histopathological grade and a most common histological type. Thus, it is difficult to predict the prognosis of RCC using existing clinicopathological factors or the like.
Analyses by MSP, COBRA, and bacterial artificial chromosome (BAC) array-based methylated CpG island amplification (BAMCA) have showed that noncancerous renal cortical tissues obtained from RCC patients are already at a precancerous stage associated with change in DNA methylation status (Patent Literature 3 and Non Patent Literatures 8 to 11). In addition, genome-wide analysis by BAMCA has also revealed that change in DNA methylation in noncancerous renal cortical tissues at a precancerous stage is inherited to the corresponding RCC in the same patients. A method for predicting the prognosis of an RCC case has been successfully developed (Patent Literature 3 and Non Patent Literature 10).
Recently, it has been found that highly malignant RCC exhibits a CIMP-positive phenotype, and methylation at the CpG sites of 17 genes (FAM150A, GRM6, ZNF540, ZFP42, ZNF154, RIMS4, PCDHAC1, KHDRBS2, ASCL2, KCNQ1, PRAC, WNT3A, TRH, FAM78A, ZNF671, SLC13A5, and NKX6-2) is a feature of CIMP of RCC (Patent Literature 4). Patent Literature 4 has proposed a method for detecting a risk of poor prognosis of RCC by detecting a methylation level at the CpG sites of those 17 genes by bead array method, mass spectrometry (MassARRAY method), pyrosequencing, methylation-sensitive high-resolution melting curve analysis, quantitative PCR, direct sequencing of bisulfite treatment products, COBRA, or the like. The value of a DNA methylation rate obtained by a method conventionally used, such as MassARRAY method or pyrosequencing, is obtained as an average DNA methylation rate of the whole sample subjected to the assay. Therefore, in the case of a sample rich in cells having a low DNA methylation rate, the obtained value of a DNA methylation rate is low even if cells having highly methylated DNA coexist therewith. This causes problems that the presence or absence of cells having highly methylated DNA cannot be determined, and a risk of estimating DNA methylation rates to be lower than actual values cannot be avoided.